1887

Abstract

Bacterial chromosomes (though not and some other -proteobacterial chromosomes) contain sequences and genes encoding partitioning proteins, i.e. ParA (ATPase) and ParB (DNA-binding proteins) that are components of the segregation machinery. Here, mycobacterial elements were characterized for the first time. genes are not essential in however, elimination or overexpression of ParB protein causes growth inhibition. Deletion of also leads to a rather severe chromosome segregation defect: up to 10 % of the cells were anucleate. Mycobacterial ParB protein uses three -proximal sequences as targets to organize the origin region into a compact nucleoprotein complex. Formation of such a complex involves ParB–ParB interactions and is assisted by ParA protein.

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2007-12-01
2019-11-12
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Oligonucleotides used in this study. [ PDF] (94 kb) Construction of the mutant The chromosomal (MSMEG_6938) and (MSMEG_6939) genes are represented by grey arrows (A). The restriction sites used for digestion of chromosomal DNA are denoted by single letters (H, III; S, I). The restriction fragment and the size of the internal deletion in the mutated copy of (black rectangle) are shown by double-headed arrows. The single-headed arrows represent PCR primers. The genomic DNA designated for PCR (B) and Southern hybridization (C) was isolated from wild-type (wt); single cross-over homologous recombination mutants ( ); double cross-over homologous recombination mutants carrying the wild-type gene ( ) or the gene with the internal deletion (ΔparB), exclusively. [ PDF] (340 kb) Purified mycobacterial ParAB proteins. The fusion proteins glutathione -transferase-ParA or 6His-ParB were purified on a glutathione-Sepharose 4B or a Ni -NTA-agarose column (Qiagen), respectively. For removal of the glutathione -transferase part, the fusion proteins bound to glutathione-Sepharose beads were treated with PreScission protease (Amersham-Pharmacia Biotech) and the ParA proteins were released from the beads. His-tagged proteins were eluted from the Ni -NTA-agarose column with 100 nM imidazole. Ms, ; Mt, . Proteins were analysed in a 12% SDS-PAGE gel and stained with Coomassie brilliant blue. [ PDF] (247 kb) Protein sequence alignment of homologous ParB proteins. Identical and similar amino acids are shown with a yellow or green background, respectively. The colours of the secondary structure elements (S, beta-sheet; H, alpha-helix) correspond to those used for SpoOJ protein by Leonard (2004). The HTH motif is coloured yellow. Identical residues in and are shown in bold type. [ PDF] (319 kb) Towards understanding the molecular basis of bacterial DNA segregation. , 523-535.

PDF

Oligonucleotides used in this study. [ PDF] (94 kb) Construction of the mutant The chromosomal (MSMEG_6938) and (MSMEG_6939) genes are represented by grey arrows (A). The restriction sites used for digestion of chromosomal DNA are denoted by single letters (H, III; S, I). The restriction fragment and the size of the internal deletion in the mutated copy of (black rectangle) are shown by double-headed arrows. The single-headed arrows represent PCR primers. The genomic DNA designated for PCR (B) and Southern hybridization (C) was isolated from wild-type (wt); single cross-over homologous recombination mutants ( ); double cross-over homologous recombination mutants carrying the wild-type gene ( ) or the gene with the internal deletion (ΔparB), exclusively. [ PDF] (340 kb) Purified mycobacterial ParAB proteins. The fusion proteins glutathione -transferase-ParA or 6His-ParB were purified on a glutathione-Sepharose 4B or a Ni -NTA-agarose column (Qiagen), respectively. For removal of the glutathione -transferase part, the fusion proteins bound to glutathione-Sepharose beads were treated with PreScission protease (Amersham-Pharmacia Biotech) and the ParA proteins were released from the beads. His-tagged proteins were eluted from the Ni -NTA-agarose column with 100 nM imidazole. Ms, ; Mt, . Proteins were analysed in a 12% SDS-PAGE gel and stained with Coomassie brilliant blue. [ PDF] (247 kb) Protein sequence alignment of homologous ParB proteins. Identical and similar amino acids are shown with a yellow or green background, respectively. The colours of the secondary structure elements (S, beta-sheet; H, alpha-helix) correspond to those used for SpoOJ protein by Leonard (2004). The HTH motif is coloured yellow. Identical residues in and are shown in bold type. [ PDF] (319 kb) Towards understanding the molecular basis of bacterial DNA segregation. , 523-535.

PDF

Oligonucleotides used in this study. [ PDF] (94 kb) Construction of the mutant The chromosomal (MSMEG_6938) and (MSMEG_6939) genes are represented by grey arrows (A). The restriction sites used for digestion of chromosomal DNA are denoted by single letters (H, III; S, I). The restriction fragment and the size of the internal deletion in the mutated copy of (black rectangle) are shown by double-headed arrows. The single-headed arrows represent PCR primers. The genomic DNA designated for PCR (B) and Southern hybridization (C) was isolated from wild-type (wt); single cross-over homologous recombination mutants ( ); double cross-over homologous recombination mutants carrying the wild-type gene ( ) or the gene with the internal deletion (ΔparB), exclusively. [ PDF] (340 kb) Purified mycobacterial ParAB proteins. The fusion proteins glutathione -transferase-ParA or 6His-ParB were purified on a glutathione-Sepharose 4B or a Ni -NTA-agarose column (Qiagen), respectively. For removal of the glutathione -transferase part, the fusion proteins bound to glutathione-Sepharose beads were treated with PreScission protease (Amersham-Pharmacia Biotech) and the ParA proteins were released from the beads. His-tagged proteins were eluted from the Ni -NTA-agarose column with 100 nM imidazole. Ms, ; Mt, . Proteins were analysed in a 12% SDS-PAGE gel and stained with Coomassie brilliant blue. [ PDF] (247 kb) Protein sequence alignment of homologous ParB proteins. Identical and similar amino acids are shown with a yellow or green background, respectively. The colours of the secondary structure elements (S, beta-sheet; H, alpha-helix) correspond to those used for SpoOJ protein by Leonard (2004). The HTH motif is coloured yellow. Identical residues in and are shown in bold type. [ PDF] (319 kb) Towards understanding the molecular basis of bacterial DNA segregation. , 523-535.

PDF

Oligonucleotides used in this study. [ PDF] (94 kb) Construction of the mutant The chromosomal (MSMEG_6938) and (MSMEG_6939) genes are represented by grey arrows (A). The restriction sites used for digestion of chromosomal DNA are denoted by single letters (H, III; S, I). The restriction fragment and the size of the internal deletion in the mutated copy of (black rectangle) are shown by double-headed arrows. The single-headed arrows represent PCR primers. The genomic DNA designated for PCR (B) and Southern hybridization (C) was isolated from wild-type (wt); single cross-over homologous recombination mutants ( ); double cross-over homologous recombination mutants carrying the wild-type gene ( ) or the gene with the internal deletion (ΔparB), exclusively. [ PDF] (340 kb) Purified mycobacterial ParAB proteins. The fusion proteins glutathione -transferase-ParA or 6His-ParB were purified on a glutathione-Sepharose 4B or a Ni -NTA-agarose column (Qiagen), respectively. For removal of the glutathione -transferase part, the fusion proteins bound to glutathione-Sepharose beads were treated with PreScission protease (Amersham-Pharmacia Biotech) and the ParA proteins were released from the beads. His-tagged proteins were eluted from the Ni -NTA-agarose column with 100 nM imidazole. Ms, ; Mt, . Proteins were analysed in a 12% SDS-PAGE gel and stained with Coomassie brilliant blue. [ PDF] (247 kb) Protein sequence alignment of homologous ParB proteins. Identical and similar amino acids are shown with a yellow or green background, respectively. The colours of the secondary structure elements (S, beta-sheet; H, alpha-helix) correspond to those used for SpoOJ protein by Leonard (2004). The HTH motif is coloured yellow. Identical residues in and are shown in bold type. [ PDF] (319 kb) Towards understanding the molecular basis of bacterial DNA segregation. , 523-535.

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